T cell receptors

ABSTRACT

The present invention relates to T cell receptors (TCRs) that bind the HLA-A*02 restricted peptide SLLQHLIGL (SEQ ID NO: 1) derived from the germline cancer antigen PRAME. Said TCRs may comprise non-natural mutations within the alpha and/or beta variable domains relative to a native PRAME TCR. The TCRs of the invention are particularly suitable for use as novel immunotherapeutic reagents for the treatment of malignant disease.

This application is a continuation of co-pending U.S. application Ser.No. 16/624,853, filed Dec. 19, 2019, which is the National Stage ofInternational Application No. PCT/EP2018/066287, filed Jun. 19, 2018,which claims the benefit of and priority to Great Britain PatentApplication Serial No. 1709866.6, filed on Jun. 20, 2017, the contentsof which are incorporated by reference in their entirety.

The present invention relates to T cell receptors (TCRs) that bind theHLA-A*02 restricted peptide SLLQHLIGL (SEQ ID NO: 1) derived from thegermline cancer antigen PRAME. Said TCRs may comprise non-naturalmutations within the alpha and/or beta variable domains relative to anative PRAME TCR. The TCRs of the invention are particularly suitablefor use as novel immunotherapeutic reagents for the treatment ofmalignant disease.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Feb. 14, 2022, is named50273US_CRF_sequencelisting.txt, and is 51,927 bytes in size

BACKGROUND TO THE INVENTION

T cell receptors (TCRs) are naturally expressed by CD4⁺ and CD8⁺ Tcells. TCRs are designed to recognize short peptide antigens that aredisplayed on the surface of antigen presenting cells in complex withMajor Histocompatibility Complex (MHC) molecules (in humans, MHCmolecules are also known as Human Leukocyte Antigens, or HLA) (Davis etal., Annu Rev Immunol. 1998; 16:523-44). CD8⁺ T cells, which are alsotermed cytotoxic T cells, have TCRs that specifically recognize peptidesbound to MHC class I molecules. CD8⁺ T cells are generally responsiblefor finding and mediating the destruction of diseased cells, includingcancerous and virally infected cells. The affinity of cancer-specificTCRs in the natural repertoire for corresponding antigen is typicallylow as a result of thymic selection, meaning that cancerous cellsfrequently escape detection and destruction. Novel immunotherapeuticapproaches aimed at promoting cancer recognition by T cells offer ahighly promising strategy for the development of effective anticancertreatments.

PRAME or Preferentially Expressed Antigen In Melanoma was firstidentified as an antigen that is over expressed in melanoma (Ikeda et alImmunity. 1997 February; 6(2):199-208); it is also known as CT130, MAPE,OIP-4 and has Uniprot accession number P78395. The protein functions asa repressor of retinoic acid receptor signalling (Epping et al., Cell.2005 Sep. 23; 122(6):835-47). PRAME belongs to the family ofgermline-encoded antigens known as cancer testis antigens. Cancer testisantigens are attractive targets for immunotherapeutic intervention sincethey typically have limited or no expression in normal adult tissues.PRAME is expressed in a number of solid tumours as well as in leukaemiasand lymphomas (Doolan et al Breast Cancer Res Treat. 2008 May;109(2):359-65; Epping et al Cancer Res. 2006 Nov. 15; 66(22):10639-42;Ercolak et al Breast Cancer Res Treat. 2008 May; 109(2):359-65;Matsushita et al Leuk Lymphoma. 2003 March; 44(3):439-44; Mitsuhashi etal Int. J Hematol. 2014; 100(1):88-95; Proto-Sequeire et al Leuk Res.2006 November; 30(11):1333-9; Szczepanski et al Oral Oncol. 2013February; 49(2):144-51; Van Baren et al Br J Haematol. 1998 September;102(5):1376-9). PRAME targeting therapies of the inventions may beparticularly suitable for treatment cancers including, but not limitedto, lung (NSCLC and SCLC), breast (including triple negative), ovarian,endometrial, oesophageal, bladder and head and neck cancers.

The peptide SLLQHLIGL (SEQ ID NO: 1) corresponds to amino acids 425-433of the full length PRAME protein and is presented on the cell surface incomplex with HLA-A*02 (Kessler et al., J Exp Med. 2001 Jan. 1;193(1):73-88). This peptide-HLA complex provides a useful target forTCR-based immunotherapeutic intervention.

The identification of particular TCR sequences that bind to theSLLQHLIGL (SEQ ID NO: 1) HLA-A*02 complex with high affinity and highspecificity is advantageous for the development of novelimmunotherapies. Therapeutic TCRs may be used, for example, as solubletargeting agents for the purpose of delivering cytotoxic agents to thetumour site or activating immune effector functions against the tumourcells (Lissin, et al., “High-Affinity Monocloncal T-cell receptor (mTCR)Fusions” in Fusion Protein Technologies for Biophamaceuticals:Applications and Challenges. 2013. S. R. Schmidt, Wiley; Boulter et al.,Protein Eng. 2003 September; 16(9):707-11; Liddy, et al., Nat Med. 2012June; 18(6):980-7), or alternatively they may be used to engineer Tcells for adoptive therapy (Fesnak et al., Nat Rev Cancer. 2016 Aug. 23;16(9):566-81).

TCRs that bind to SLLQHLIGL (SEQ ID NO: 1) in complex with HLA-A*02 havebeen reported previously (Amir et al., Clin Cancer Res. 2011 Sep. 1;17(17):5615-25; Griffioen et al., Clin Cancer Res. 2006 May 15;12(10):3130-6; WO2016142783). However, these TCRs have not beenengineered so that they bind to the target antigen with increasedaffinity, relative to the natural TCR. As explained further below,supra-physiological antigen affinity is a desirable feature for atherapeutic TCR, the production of which is not straightforward,particularly when balanced with other desirable features, such asspecificity.

The TCR sequences defined herein are described with reference to IMGTnomenclature which is widely known and accessible to those working inthe TCR field. For example, see: LeFranc and LeFranc, (2001). “T cellReceptor Factsbook”, Academic Press; Lefranc, (2011), Cold Spring HarbProtoc 2011(6): 595-603; Lefranc, (2001), Curr Protoc Immunol Appendix1: Appendix 100; and Lefranc, (2003), Leukemia 17(1): 260-266. Briefly,αβ TCRs consist of two disulphide linked chains. Each chain (alpha andbeta) is generally regarded as having two domains, namely a variable anda constant domain. A short joining region connects the variable andconstant domains and is typically considered part of the alpha variableregion. Additionally, the beta chain usually contains a short diversityregion next to the joining region, which is also typically consideredpart of the beta variable region.

The variable domain of each chain is located N-terminally and comprisesthree Complementarity Determining Regions (CDRs) embedded in a frameworksequence (FR). The CDRs comprise the recognition site for peptide-MHCbinding. There are several genes coding for alpha chain variable (Vα)regions and several genes coding for beta chain variable (Vβ) regions,which are distinguished by their framework, CDR1 and CDR2 sequences, andby a partly defined CDR3 sequence. The Vα and Vβ genes are referred toin IMGT nomenclature by the prefix TRAV and TRBV respectively (Folch andLefranc, (2000), Exp Clin Immunogenet 17(1): 42-54; Scaviner andLefranc, (2000), Exp Clin Immunogenet 17(2): 83-96; LeFranc and LeFranc,(2001), “T cell Receptor Factsbook”, Academic Press). Likewise there areseveral joining or J genes, termed TRAJ or TRBJ, for the alpha and betachain respectively, and for the beta chain, a diversity or D gene termedTRBD (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(2): 107-114;Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2): 97-106;LeFranc and LeFranc, (2001), “T cell Receptor Factsbook”, AcademicPress). The huge diversity of T cell receptor chains results fromcombinatorial rearrangements between the various V, J and D genes, whichinclude allelic variants, and junctional diversity (Arstila, et al.,(1999), Science 286(5441): 958-961; Robins et al., (2009), Blood114(19): 4099-4107.) The constant, or C, regions of TCR alpha and betachains are referred to as TRAC and TRBC respectively (Lefranc, (2001),Curr Protoc Immunol Appendix 1: Appendix 10).

The inventors of the present application have surprisingly found novelTCRs that are able to bind to SLLQHLIGL-HLA-A*02 complex (“SLLQHLIGL”disclosed as SEQ ID NO: 1) with high affinity and specificity. Said TCRsare engineered from a suitable scaffold sequence into which a number ofmutations are introduced. The TCRs of the invention have a particularlysuitable profile for therapeutic use. In general, the identification ofsuch TCRs is not straightforward and typically has a high attritionrate.

In the first instance, the skilled person needs to identify a suitablestarting, or scaffold, sequence. Typically such sequences are obtainedfrom natural sources e.g. from antigen responding T cells extracted fromdonor blood. Given the rarity of cancer specific T cells in the naturalrepertoire, it is often necessary to screen many donors, for example 20or more, before a responding T cell may be found. The screening processmay take several weeks or months, and even where a responding T cell isfound, it may be unsuitable for immunotherapeutic use. For example, theresponse may be too weak and/or may not be specific for the targetantigen. Alternatively, it may not be possible to generate a clonal Tcell population, nor expand or maintain a given T cell line to producesufficient material to identify the correct TCR chain sequences. TCRsequences that are suitable as starting, or scaffold, sequences shouldhave one or more of the following properties: a good affinity for thetarget peptide-HLA complex, for example 200 μM or stronger; a high levelof target specificity, e.g. relatively weak or no binding to alternativepeptide-HLA complexes; be amenable to use in display libraries, such asphage display; and be able to be refolded and purified at high yield.Given the degenerate nature of TCR recognition, it is exceptionally hardeven for skilled practitioners to be able to determine whether aparticular scaffold TCR sequence has a specificity profile that wouldmake it eligible for engineering for therapeutic use (Wooldridge, etal., J Biol Chem. 2012 Jan. 6; 287(2):1168-77).

The next challenge is to engineer the TCR to have a higher affinitytowards the target antigen whilst retaining desirable characteristicssuch as specificity and yield. TCRs, as they exist in nature, have weakaffinity for target antigen (low micromolar range) compared withantibodies, and TCRs against cancer antigens typically have weakerantigen recognition than viral specific TCRs (Aleksic, et al. Eur JImmunol. 2012 December; 42(12):3174-9). This weak affinity coupled withHLA down-regulation on cancer cells means that therapeutic TCRs forcancer immunotherapy typically require engineering to increase theiraffinity for target antigen and thus generate a more potent response.Such affinity increases are essential for soluble TCR-based reagents. Insuch cases, antigen-binding affinities in the nanomolar to picomolarrange, with binding half-lives of several hours, are desirable. Theimproved potency generated by high affinity antigen recognition at lowepitope numbers is exemplified in FIGS. 1e and 1f of Liddy et al.(Liddy, et al., Nat Med. 2012 June; 18(6):980-7). The affinitymaturation process, typically involves the skilled person having toengineer specific mutations and/or combinations of mutations, includingbut not limited to substitutions, insertions and/or deletions, on to thestarting TCR sequence in order to increase the strength of antigenrecognition. Methods to engineer affinity enhancing mutations on to agiven TCR are known in the art, for example the use of display libraries(Li et al., Nat Biotechnol. 2005 March; 23(3):349-54; Holler et al.,Proc Natl Acad Sci USA. 2000 May 9; 97(10):5387-92). However, to producesignificant increases in the affinity of a given TCR against a giventarget, the skilled person may have to engineer combinations ofmutations from a large pool of possible alternatives. The specificmutations and/or combinations of mutations that produce significantincreases in affinity are not predictable and there is a high attritionrate. In many cases, it may not be possible to achieve significantincreases in affinity with a given TCR starting sequence.

The affinity maturation process must also take account of the necessityof maintaining TCR antigen specificity. Increasing the affinity of a TCRfor its target antigen brings a substantial risk of revealing crossreactivity with other unintended targets as a result of the inherentdegeneracy of TCR antigen recognition (Wooldridge, et al., J Biol Chem.2012 Jan. 6; 287(2):1168-77; Wilson, et al., Mol Immunol. 2004 February;40(14-15):1047-55; Zhao et al., J Immunol. 2007 Nov. 1; 179(9):5845-54).At a natural level of affinity the recognition of the cross reactiveantigen may be too low to produce a response. If a cross reactiveantigen is displayed on normal healthy cells, there is a strongpossibility of off-target binding in vivo which may manifest in clinicaltoxicity. Thus, in addition to increasing antigen binding strength, theskilled person must also engineer mutations and or combinations ofmutations that allow the TCR to retain a high specificity for targetantigen and demonstrate a good safety profile in preclinical testing.Again, suitable mutations and/or combinations of mutations are notpredictable. The attrition rate at this stage is even higher and in manycases may not be achievable at all from a given TCR starting sequence.

Despite the difficulties described above, the inventors have identifiedmutated TCRs with a particularly high affinity (picomolar range), and ahigh degree of antigen specificity. Said TCRs demonstrate potent killingof PRAME positive cancer cells when prepared as soluble reagents fusedto a T cell redirecting moiety.

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides a T cell receptor(TCR) having the property of binding to SLLQHLIGL (SEQ ID NO: 1) incomplex with HLA-A*02 and comprising a TCR alpha chain variable domainand/or a TCR beta chain variable domain, each of which comprisesFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 where FR is a framework region and CDR isa complementarity determining region, wherein

-   -   (a) the alpha chain CDRs have the following sequences:        -   CDR1—TISGTDY(SEQ ID NO: 39)        -   CDR2—GLTSN (SEQ ID NO: 40)        -   CDR3—CILILGHSGAGSYQLTF(SEQ ID NO: 41)    -   optionally with one or more mutations therein,        and/or    -   (b) the beta chain CDRs have the following sequences:        -   CDR1—LNHDA (SEQ ID NO: 42)        -   CDR2—SQIVNDF (SEQ ID NO: 43)        -   CDR3—CASSPWTSGSREQYF (SEQ ID NO: 44)    -   optionally with one or more mutations therein.

In the TCR of the first aspect, the alpha chain variable domainframework regions may comprise the following framework sequences:

-   -   FR1—amino acids 1-25 of SEQ ID NO: 2    -   FR2—amino acids 33-49 of SEQ ID NO: 2    -   FR3—amino acids 55-87 of SEQ ID NO: 2    -   FR4—amino acids 105-114 of SEQ ID NO: 2    -   or respective sequences having at least 90, 91, 92, 93, 94, 95,        96, 97, 98 or 99% identity to said sequences, and/or        the beta chain variable domain framework regions may comprise        the following sequences:    -   FR1—amino acids 1-26 of SEQ ID NO: 3    -   FR2—amino acids 32-48 of SEQ ID NO: 3    -   FR3—amino acids 56-90 of SEQ ID NO: 3    -   FR4—amino acids 106-114 of SEQ ID NO: 3    -   or respective sequences having at least 90, 91, 92, 93, 94, 95,        96, 97, 98 or 99% identity to said sequences.

The term ‘mutations’ encompasses substitutions, insertions anddeletions. Mutations to a parental (or wild type, or scaffold) TCR mayinclude those that increase the binding affinity (k_(D) and/or bindinghalf life) of the TCR to SLLQHLIGL-HLA-A*02 complex (“SLLQHLIGL”disclosed as SEQ ID NO: 1).

Conventionally, beta chain residue F55 is considered to be part offramework region 3. However, for the purposes of the present invention,beta chain residue F55 is considered part of CDR2.

The alpha chain framework regions FR1, FR2, and FR3 may comprise aminoacid sequences corresponding to a TRAV 26-2 chain and/or the beta chainframework regions FR1, FR2 and FR3, may comprise amino acid sequencescorresponding to those of a TRBV19 chain.

The FR4 region may comprise the joining region of the alpha and betavariable chains (TRAJ and TRBJ, respectively).

In the TCR alpha chain variable region, there may be at least onemutation. There may be one, two, three, four or five, or more, mutationsin the alpha chain CDRs. There may be one, two, three, four or fivemutations in the alpha chain CDR3. One or more of said mutations may beselected from the following mutations, with reference to the numberingof SEQ ID NO: 2:

Wild type Mutation G96 (CDR3) R A97 (CDR3) L S99 (CDR3) N Q101 (CDR3) IL102 (CDR3) A

Thus, there may be any or all of the mutations in the table above,optionally in combination with other mutations.

The alpha chain CDR3 may comprise one of the following groups ofmutations (with reference to the numbering of SEQ ID NO: 2):

1 G96R S99N Q101I L102A 2 G96R A97L S99N Q101I L102A 3 G96R A97L S99NL102A

A preferred group of mutations is group 1. Another preferred group ofmutations is group 2.

The alpha chain CDR3 may have a sequence selected from:

(SEQ ID NO: 45) CILILGHSRAGNYIATF (SEQ ID NO: 46) CILILGHSRLGNYIATF(SEQ ID NO: 47) CILILGHSRLGNYQATF

A preferred alpha chain CDR3 is CILILGHSRAGNYIATF (SEQ ID NO: 45). Apreferred alpha chain CDR3 is CILILGHSRLGNYIATF (SEQ ID NO: 46).

In the TCR beta chain variable region, there may be at least onemutation. There may be one, two, three, four, five, six, seven, eight,nine 10, or more, mutations in the beta chain CDRs. There may be one,two, three, four, five, six, seven, eight, nine or ten mutations in thebeta chain CDR3. One or more of said mutations may be selected from thefollowing mutations with reference to the numbering of SEQ ID NO: 3:

Wild type Mutation V52 (CDR2) M N53 (CDR2) G F55 (CDR2) E P95 (CDR3) WS98 (CDR3) G S100 (CDR3) A R101 (CDR3) S or A E102 (CDR3) P Q103 (CDR3)I Y104 (CDR3) S or R

Thus, there may be any or all of the mutations in the table above,optionally in combination with other mutations.

The beta chain CDR2 and CDR3 may comprise one of the following groups ofmutations (with reference to the numbering of SEQ ID NO: 3):

1 V52M N53G F55E P95W S98G S100A R101S E102P Q103I Y104S 2 V52M N53GF55E P95W S100A R101S E102P Q103I Y104S 3 V52M N53G F55E P95W S98G R101AE102P Q103I Y104R 4 V52M F55E P95W S98G R101A E102P Q103I Y104R 5 V52MN53G F55E P95W R101A E102P Q103I Y104R 6 V52M N53G F55E P95W S98G R101AQ103I Y104R 7 V52M N53G F55E P95W S98G R101A E102P Q103I 8 V52M N53GF55E P95W S98G S100A R101A E102P Q103I Y104S 9 V52M N53G F55E P95W S98GS100A R101S E102P Q103I Y104R 10 V52M N53G F55E P95W S98G S100A R101AE102P Q103I Y104R 11 V52M N53G F55E P95W S98G S100A R101S Q103I Y104S 12V52M N53G F55E P95W S98G R101A E102P Q103I Y104S 13 V52M N53G F55E P95WS98G R101S E102P Q103I Y104S 14 N53G F55E P95W S98G R101A E102P Q103IY104R 15 V52M N53G F55E S98G R101A E102P Q103I Y104R 16 V52M N53G F55EP95W S98G R101S E102P Q103I Y104R

A preferred group of mutations is group 1. A preferred group ofmutations is group 9.

The beta chain CDR2 may have a sequence selected from:

(SEQ ID NO: 48) SQIMGDE (SEQ ID NO: 49) SQIMNDE (SEQ ID NO: 50) SQIVGDE

A preferred beta chain CDR2 is SQIMGDE (SEQ ID NO: 48).

The beta chain CDR3 may have a sequence selected from:

(SEQ ID NO: 51) CASSWWTGGASPISF (SEQ ID NO: 52) CASSWWTSGASPISF(SEQ ID NO: 53) CASSWWTGGSAPIRF (SEQ ID NO: 54) CASSWWTSGSAPIRF(SEQ ID NO: 55) CASSWWTGGSAEIRF (SEQ ID NO: 56) CASSWWTGGSAPIYF(SEQ ID NO: 57) CASSWWTGGAAPISF (SEQ ID NO: 58) CASSWWTGGASPIRF(SEQ ID NO: 59) CASSWWTGGAAPIRF (SEQ ID NO: 60) CASSWWTGGASEISF(SEQ ID NO: 61) CASSWWTGGSAPISF (SEQ ID NO: 62) CASSWWTGGSSPISF(SEQ ID NO: 63) CASSPWTGGSAPIRF (SEQ ID NO: 64) CASSWWTGGSSPIRF

A preferred beta chain CDR3 is CASSWWTGGASPISF (SEQ ID NO: 51). Apreferred beta chain CDR3 is CASSWWTGGASPIRF (SEQ ID NO: 58).

Preferred combinations of beta chain CDR2 and CDR3 are as follows:

 1 SQIMGDE (SEQ ID NO: 48) CASSWWTGGASPISF (SEQ ID NO: 51)  2SQIMGDE (SEQ ID NO: 48) CASSWWTSGASPISF (SEQ ID NO: 52)  3SQIMGDE (SEQ ID NO: 48) CASSWWTGGSAPIRF (SEQ ID NO: 53)  4SQIMNDE (SEQ ID NO: 49) CASSWWTGGSAPIRF (SEQ ID NO: 53)  5SQIMGDE (SEQ ID NO: 48) CASSWWTSGSAPIRF (SEQ ID NO: 54)  6SQIMGDE (SEQ ID NO: 48) CASSWWTGGSAEIRF (SEQ ID NO: 55)  7SQIMGDE (SEQ ID NO: 48) CASSWWTGGSAPIYF (SEQ ID NO: 56)  8SQIMGDE (SEQ ID NO: 48) CASSWWTGGAAPISF (SEQ ID NO: 57)  9SQIMGDE (SEQ ID NO: 48) CASSWWTGGASPIRF (SEQ ID NO: 58) 10SQIMGDE (SEQ ID NO: 48) CASSWWTGGAAPIRF (SEQ ID NO: 59) 11SQIMGDE (SEQ ID NO: 48) CASSWWTGGASEISF (SEQ ID NO: 60) 12SQIMGDE (SEQ ID NO: 48) CASSWWTGGSAPISF (SEQ ID NO: 61) 13SQIMGDE (SEQ ID NO: 48) CASSWWTGGSSPISF (SEQ ID NO: 62) 14SQIVGDE (SEQ ID NO: 50) CASSWWTGGSAPIRF (SEQ ID NO: 53) 15SQIMGDE (SEQ ID NO: 48) CASSPWTGGSAPIRF (SEQ ID NO: 63) 16SQIMGDE (SEQ ID NO: 48) CASSWWTGGSSPIRF (SEQ ID NO: 64)

A preferred combination is combination 1. Another preferred combinationis combination 9.

In a preferred embodiment, the TCR alpha and beta chain CDR sequencesare selected from:

Alpha Beta CDR1 CDR2 CDR3 CDR1 CDR2 CDR3  1 TISGTDY GLTSNCILILGHSRAGNYIATF LNHDA SQIMGDE CASWWTGGASPISF (SEQ ID (SEQ ID NO: 40)(SEQ ID NO: 45) (SEQ ID (SEQ ID NO: (SEQ ID NO: 51) NO: 39) NO: 42) 48) 2 TISGTDY GLTSN CILILGHSRAGNYIATF LNHDA SQIMGDE CASSWWTGGASEISF (SEQ ID(SEQ ID NO: 40) (SEQ ID NO: 45) (SEQ ID (SEQ ID NO: (SEQ ID NO: 60)NO: 39) NO: 42) 48)  3 TISGTDY GLTSN CILILGHSRAGNYIATF LNHDA SQIMGDECASSWWTGGASPIRF (SEQ ID (SEQ ID NO: 40) (SEQ ID NO: 45) (SEQ ID(SEQ ID NO: (SEQ ID NO: 58) NO: 39) NO: 42) 48)  4 TISGTDY GLTSNCILILGHSRAGNYIATF LNHDA SQIMGDE CASSWWTGGSAPISF (SEQ ID (SEQ ID NO: 40)(SEQ ID NO: 45) (SEQ ID (SEQ ID NO: (SEQ ID NO: 61) NO: 39) NO: 42) 48) 5 TISGTDY GLTSN CILILGHSRAGNYIATF LNHDA SQIMGDE CASSWWTSGASPISF (SEQ ID(SEQ ID NO: 40) (SEQ ID NO: 45) (SEQ ID (SEQ ID NO: (SEQ ID NO: 52)NO: 39) NO: 42) 48)  6 TISGTDY GLTSN CILILGHSRAGNYIATF LNHDA SQIMGDECASSWWTGGSSPISF (SEQ ID (SEQ ID NO: 40) (SEQ ID NO: 45) (SEQ ID(SEQ ID NO: (SEQ ID NO: 62) NO: 39) NO: 42) 48)  7 TISGTDY GLTSNCILILGHSRLGNYIATF LNHDA SQIMGDE CASSWWTGGSAPIRF (SEQ ID (SEQ ID NO: 40)(SEQ ID NO: 46) (SEQ ID (SEQ ID NO: (SEQ ID NO: 53) NO: 39) NO: 42) 48) 8 TISGTDY GLTSN CILILGHSRLGNYQATF LNHDA SQIMGDE CASSWWTGGSAPIRF (SEQ ID(SEQ ID NO: 40) (SEQ ID NO: 47) (SEQ ID (SEQ ID NO: (SEQ ID NO: 53)NO: 39) NO: 42) 48)  9 TISGTDY GLTSN CILILGHSRLGNYIATF LNHDA SQIVGDECASSWWTGGSAPIRF (SEQ ID (SEQ ID NO: 40) (SEQ ID NO: 46) (SEQ ID(SEQ ID NO: (SEQ ID NO: 53) NO: 39) NO: 42) 50) 10 TISGTDY GLTSNCILILGHSRLGNYIATF LNHDA SQIMNDE CASSWWTGGSAPIRF (SEQ ID (SEQ ID NO: 40)(SEQ ID NO: 46) (SEQ ID (SEQ ID NO: (SEQ ID NO: 53) NO: 39) NO: 42) 49)11 TISGTDY GLTSN CILILGHSRLGNYIATF LNHDA SQIMGDE CASSPWTGGSAPIRF (SEQ ID(SEQ ID NO: 40) (SEQ ID NO: 46) (SEQ ID (SEQ ID NO: (SEQ ID NO: 63)NO: 39) NO: 42) 48) 12 TISGTDY GLTSN CILILGHSRLGNYIATF LNHDA SQIMGDECASSWWTSGSAPIRF (SEQ ID (SEQ ID NO: 40) (SEQ ID NO: 46) (SEQ ID(SEQ ID NO: (SEQ ID NO: 54) NO: 39) NO: 42) 48) 13 TISGTDY GLTSNCILILGHSRLGNYIATF LNHDA SQIMGDE CASSWWTGGSAEIRF (SEQ ID (SEQ ID NO: 40)(SEQ ID NO: 46) (SEQ ID (SEQ ID NO: (SEQ ID NO: 55) NO: 39) NO: 42) 48)14 TISGTDY GLTSN CILILGHSRLGNYIATF LNHDA SQIMGDE CASSWWTGGSAPIYF (SEQ ID(SEQ ID NO: 40) (SEQ ID NO: 46) (SEQ ID (SEQ ID NO: (SEQ ID NO: 56)NO: 39) NO: 42) 48) 15 TISGTDY GLTSN CILILGHSRLGNYIATF LNHDA SQIMGDECASSWWTGGSSPISF (SEQ ID (SEQ ID NO: 40) (SEQ ID NO: 46) (SEQ ID(SEQ ID NO: (SEQ ID NO: 62) NO: 39) NO: 42) 48) 16 TISGTDY GLTSNCILILGHSRLGNYIATF LNHDA SQIMGDE CASSWWTGGAAPISF (SEQ ID (SEQ ID NO: 40)(SEQ ID NO: 46) (SEQ ID (SEQ ID NO: (SEQ ID NO: 57) NO: 39) NO: 42) 48)17 TISGTDY GLTSN CILILGHSRLGNYIATF LNHDA SQIMGDE CASSWWTGGASPIRF (SEQ ID(SEQ ID NO: 40) (SEQ ID NO: 46) (SEQ ID (SEQ ID NO: (SEQ ID NO: 58)NO: 39) NO: 42) 48) 18 TISGTDY GLTSN CILILGHSRLGNYIATF LNHDA SQIMGDECASSWTGGSAPISF (SEQ ID (SEQ ID NO: 40) (SEQ ID NO: 46) (SEQ ID(SEQ ID NO: (SEQ ID NO: 61) NO: 39) NO: 42) 48) 19 TISGTDY GLTSNCILILGHSRLGNYIATF LNHDA SQIMGDE CASSWTGGSSPIRF (SEQ ID (SEQ ID NO: 40)(SEQ ID NO: 46) (SEQ ID (SEQ ID NO: (SEQ ID NO: 64) NO: 39) NO: 42) 48)20 TISGTDY GLTSN CILILGHSRLGNYIATF LNHDA SQIMGDE CASSWTGGAAPIRF (SEQ ID(SEQ ID NO: 40) (SEQ ID NO: 46) (SEQ ID (SEQ ID NO: (SEQ ID NO: 59)NO: 39) NO: 42) 48)

A preferred combination is combination 1. A preferred combination iscombination 17.

Mutation(s) within the CDRs preferably improve the binding affinity ofthe TCR to the SLLQHLIGL-HLA-A*02 complex (“SLLQHLIGL” disclosed as SEQID NO: 1), but may additionally or alternatively confer other advantagessuch as improved stability in an isolated form and improved specificity.Mutations at one or more positions may additionally or alternativelyaffect the interaction of an adjacent position with the cognate pMHCcomplex, for example by providing a more favourable angle forinteraction. Mutations may include those that are able to reduce theamount of non-specific binding, i.e. reduce binding to alternativeantigens relative to SLLQHLIGL-HLA-A*02 (“SLLQHLIGL” disclosed as SEQ IDNO: 1). Mutations may include those that increase efficacy of foldingand/or manufacture. Some mutations may contribute to each of thesecharacteristics; others may contribute to affinity but not tospecificity, for example, or to specificity but not to affinity etc.

Typically, at least 5, at least 10, at least 15, or more CDR mutationsin total are needed to obtain TCRs with pM affinity for target antigen.At least 5, at least 10 or at least 15 CDR mutations in total may beneeded to obtain TCRs with pM affinity for target antigen. TCRs with pMaffinity for target antigen are especially suitable as solubletherapeutics. TCRs for use in adoptive therapy applications may havelower affinity for target antigen and thus fewer CDR mutations, forexample, up to 1, up to 2, up to 5, or more CDR mutations in total. TCRsfor use in adoptive therapy applications may have lower affinity fortarget antigen and thus fewer CDR mutations, for example, up to 1, up to2 or up to 5 CDR mutations in total.

Mutations may additionally, or alternatively, be made outside of theCDRs, within the framework regions; such mutations may improve binding,and/or specificity, and/or stability, and/or the yield of a purifiedsoluble form of the TCR. For example, the TCR of the invention may,additionally or alternatively, comprise an alpha chain variable domain,wherein the alpha chain variable region FR1 has a G residue at position−1 using the numbering of SEQ ID NO: 2, i.e. inserted before position 1.It was found that a G at position −1 improves cleavage efficiency of theN-terminal methionine during production in E. coli. Inefficient cleavagemay be detrimental for a therapeutic, since it may result in aheterogeneous protein product, and or the presence of the initiationmethionine may be immunogenic in humans.

Preferably, the a chain variable domain of the TCR of the invention maycomprise respective framework amino acid sequences that have at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the framework amino acid residues 1-25, 33-49, 55-87, 105-114 of SEQID NO: 2. The beta chain variable domain of the TCR of the invention maycomprise respective framework amino acid sequences that have at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the framework amino acid residues 1-26, 32-48, 56-90, 106-114 of SEQID NO: 3. Alternatively, the stated percentage identity may be over theframework sequences when considered as a whole.

The alpha chain variable domain may comprise any one of the amino acidsequences of SEQ ID NOs: 6-8 and the beta chain variable domain maycomprise any one of the amino acid sequences of SEQ ID NOs: 9-24.

For example, the TCR may comprise the following alpha and beta chainpairs.

Alpha chain Beta chain variable domain variable domain SEQ ID NO: 6 SEQID NO: 9 SEQ ID NO: 6 SEQ ID NO: 19 SEQ ID NO: 6 SEQ ID NO: 17 SEQ IDNO: 6 SEQ ID NO: 20 SEQ ID NO: 6 SEQ ID NO: 10 SEQ ID NO: 6 SEQ ID NO:21 SEQ ID NO: 7 SEQ ID NO: 11 SEQ ID NO: 8 SEQ ID NO: 11 SEQ ID NO: 7SEQ ID NO: 22 SEQ ID NO: 7 SEQ ID NO: 12 SEQ ID NO: 7 SEQ ID NO: 23 SEQID NO: 7 SEQ ID NO: 13 SEQ ID NO: 7 SEQ ID NO: 14 SEQ ID NO: 7 SEQ IDNO: 15 SEQ ID NO: 7 SEQ ID NO: 21 SEQ ID NO: 7 SEQ ID NO: 16 SEQ ID NO:7 SEQ ID NO: 17 SEQ ID NO: 7 SEQ ID NO: 20 SEQ ID NO: 7 SEQ ID NO: 24SEQ ID NO: 7 SEQ ID NO: 18

A preferred TCR chain pairing is SEQ ID NO: 6 and SEQ ID NO: 9. Apreferred TCR chain pairing is SEQ ID NO: 7 and SEQ ID NO: 17.

Within the scope of the invention are phenotypically silent variants ofany TCR of the invention disclosed herein. As used herein the term“phenotypically silent variants” is understood to refer to a TCRvariable domain which incorporates one or more further amino acidchanges, including substitutions, insertions and deletions, in additionto those set out above, which TCR has a similar phenotype to thecorresponding TCR without said change(s). For the purposes of thisapplication, TCR phenotype comprises binding affinity (K_(D) and/orbinding half-life) and specificity. Preferably, the phenotype for asoluble TCR associated with an immune effector includes potency ofimmune activation and purification yield, in addition to bindingaffinity and specificity. A phenotypically silent variant may have aK_(D) and/or binding half-life for the SLLQHLIGL-HLA-A*02 complex(“SLLQHLIGL” disclosed as SEQ ID NO: 1) within 50%, or more preferablywithin 30%, 25% or 20%, of the measured K_(D) and/or binding half-lifeof the corresponding TCR without said change(s), when measured underidentical conditions (for example at 25° C. and/or on the same SPRchip). Suitable conditions are further provided in Example 3. As isknown to those skilled in the art, it may be possible to produce TCRsthat incorporate changes in the variable domains thereof compared tothose detailed above without altering the affinity of the interactionwith the SLLQHLIGL-HLA-A*02 complex (“SLLQHLIGL” disclosed as SEQ ID NO:1), and or other functional characteristics. In particular, such silentmutations may be incorporated within parts of the sequence that areknown not to be directly involved in antigen binding (e.g. the frameworkregions and or parts of the CDRs that do not contact the antigen). Suchvariants are included in the scope of this invention.

Phenotypically silent variants may contain one or more conservativesubstitutions and/or one or more tolerated substitutions. By toleratedsubstitutions it is meant those substitutions which do not fall underthe definition of conservative as provided below but are nonethelessphenotypically silent. The skilled person is aware that various aminoacids have similar properties and thus are ‘conservative’. One or moresuch amino acids of a protein, polypeptide or peptide can often besubstituted by one or more other such amino acids without eliminating adesired activity of that protein, polypeptide or peptide.

Thus the amino acids glycine, alanine, valine, leucine and isoleucinecan often be substituted for one another (amino acids having aliphaticside chains). Of these possible substitutions it is preferred thatglycine and alanine are used to substitute for one another (since theyhave relatively short side chains) and that valine, leucine andisoleucine are used to substitute for one another (since they havelarger aliphatic side chains which are hydrophobic). Other amino acidswhich can often be substituted for one another include: phenylalanine,tyrosine and tryptophan (amino acids having aromatic side chains);lysine, arginine and histidine (amino acids having basic side chains);aspartate and glutamate (amino acids having acidic side chains);asparagine and glutamine (amino acids having amide side chains); andcysteine and methionine (amino acids having sulphur containing sidechains). It should be appreciated that amino acid substitutions withinthe scope of the present invention can be made using naturally occurringor non-naturally occurring amino acids. For example, it is contemplatedherein that the methyl group on an alanine may be replaced with an ethylgroup, and/or that minor changes may be made to the peptide backbone.Whether or not natural or synthetic amino acids are used, it ispreferred that only L-amino acids are present.

Substitutions of this nature are often referred to as “conservative” or“semi-conservative” amino acid substitutions. The present inventiontherefore extends to use of a TCR comprising any of the amino acidsequence described above but with one or more conservative substitutionsand or one or more tolerated substitutions in the sequence, such thatthe amino acid sequence of the TCR has at least 90% identity, such as90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, tothe TCR comprising amino acids 1-114 of SEQ ID NOs: 2, 6-8, and/or aminoacids 1-114 of SEQ ID NOs: 3, 9-24.

Identity” as known in the art is the relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, identity also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. While there exist a number of methods tomeasure identity between two polypeptide or two polynucleotidesequences, methods commonly employed to determine identity are codifiedin computer programs. Preferred computer programs to determine identitybetween two sequences include, but are not limited to, GCG programpackage (Devereux, et al., Nucleic Acids Research, 12, 387 (1984),BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403(1990)).

One can use a program such as the CLUSTAL program to compare amino acidsequences. This program compares amino acid sequences and finds theoptimal alignment by inserting spaces in either sequence as appropriate.It is possible to calculate amino acid identity or similarity (identityplus conservation of amino acid type) for an optimal alignment. Aprogram like BLASTx will align the longest stretch of similar sequencesand assign a value to the fit. It is thus possible to obtain acomparison where several regions of similarity are found, each having adifferent score. Both types of identity analysis are contemplated in thepresent invention.

The percent identity of two amino acid sequences or of two nucleic acidsequences is determined by aligning the sequences for optimal comparisonpurposes (e.g., gaps can be introduced in the first sequence for bestalignment with the sequence) and comparing the amino acid residues ornucleotides at corresponding positions. The “best alignment” is analignment of two sequences which results in the highest percentidentity. The percent identity is determined by the number of identicalamino acid residues or nucleotides in the sequences being compared(i.e., % identity=number of identical positions/total number ofpositions×100).

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm known to those of skill inthe art. An example of a mathematical algorithm for comparing twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. The BLASTn and BLASTp programsof Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporatedsuch an algorithm. Determination of percent identity between twonucleotide sequences can be performed with the BLASTn program.Determination of percent identity between two protein sequences can beperformed with the BLASTp program. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilised as described inAltschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively,PSI-Blast can be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilising BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., BLASTp and BLASTp) can be used. Seehttp://www.ncbi.nlm.nih.gov. Default general parameters may include forexample, Word Size=3, Expect Threshold=10. Parameters may be selected toautomatically adjust for short input sequences. Another example of amathematical algorithm utilised for the comparison of sequences is thealgorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version2.0) which is part of the CGC sequence alignment software package hasincorporated such an algorithm. Other algorithms for sequence analysisknown in the art include ADVANCE and ADAM as described in Torellis andRobotti (1994) Comput. Appl. Biosci., 10:3-5; and FASTA described inPearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. WithinFASTA, ktup is a control option that sets the sensitivity and speed ofthe search. For the purposes of evaluating percent identity in thepresent disclosure, BLASTp with the default parameters is used as thecomparison methodology. In addition, when the recited percent identityprovides a non-whole number value for amino acids (i.e., a sequence of25 amino acids having 90% sequence identity provides a value of “22.5”,the obtained value is rounded down to the next whole number, thus “22”).Accordingly, in the example provided, a sequence having 22 matches outof 25 amino acids is within 90% sequence identity.

As will be obvious to those skilled in the art, it may be possible totruncate, or extend, the sequences provided at the C-terminus and/orN-terminus thereof, by 1, 2, 3, 4, 5 or more residues, withoutsubstantially affecting the functional characteristics of the TCR. Thesequences provided at the C-terminus and/or N-terminus thereof may betruncated or extended by 1, 2, 3, 4 or 5 residues. All such variants areencompassed by the present invention.

Mutations, including conservative and tolerated substitutions,insertions and deletions, may be introduced into the sequences providedusing any appropriate method including, but not limited to, those basedon polymerase chain reaction (PCR), restriction enzyme-based cloning, orligation independent cloning (LIC) procedures. These methods aredetailed in many of the standard molecular biology texts. For furtherdetails regarding polymerase chain reaction (PCR) and restrictionenzyme-based cloning, see Sambrook & Russell, (2001) Molecular Cloning—ALaboratory Manual (3^(rd) Ed.) CSHL Press. Further information onligation independent cloning (LIC) procedures can be found inRashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6. The TCR sequencesprovided by the invention may be obtained from solid state synthesis, orany other appropriate method known in the art.

The TCRs of the invention have the property of binding theSLLQHLIGL-HLA-A*02 complex (“SLLQHLIGL” disclosed as SEQ ID NO: 1). TCRsof the invention demonstrate a high degree of specificity forSLLQHLIGL-HLA-A*02 complex (“SLLQHLIGL” disclosed as SEQ ID NO: 1) andare thus particularly suitable for therapeutic use. Specificity in thecontext of TCRs of the invention relates to their ability to recogniseHLA-A*02 target cells that are antigen positive, whilst having minimalability to recognise HLA-A*02 target cells that are antigen negative.

Specificity can be measured in vitro, for example, in cellular assayssuch as those described in Examples 6, 7 and 8. To test specificity, theTCRs may be in soluble form and associated with an immune effector,and/or may be expressed on the surface of cells, such as T cells.Specificity may be determined by measuring the level of T cellactivation in the presence of antigen positive and antigen negativetarget cells. Minimal recognition of antigen negative target cells isdefined as a level of T cell activation of less than 20%, preferablyless than 10%, preferably less than 5%, and more preferably less than1%, of the level produced in the presence of antigen positive targetcells, when measured under the same conditions and at a therapeuticallyrelevant TCR concentration. For soluble TCRs associated with an immuneeffector, a therapeutically relevant concentration may be defined as aTCR concentration of 10⁻⁹ M or below, and/or a concentration of up to100, preferably up to 1000, fold greater than the corresponding EC50value. Preferably, for soluble TCRs associated with an immune effectorthere is at least a 100 fold difference in concentration required for Tcell activation against antigen positive cells relative to antigennegative cells. Antigen positive cells may be obtained bypeptide-pulsing using a suitable peptide concentration to obtain a levelof antigen presentation comparable to cancer cells (for example, 10⁻⁹ Mpeptide, as described in Bossi et al., (2013) Oncoimmunol. 1; 2(11):e26840) or, they may naturally present said peptide. Preferably,both antigen positive and antigen negative cells are human cells.Preferably antigen positive cells are human cancer cells. Antigennegative cells preferably include those derived from healthy humantissues.

Specificity may additionally, or alternatively, relate to the ability ofa TCR to bind to SLLQHLIGL (SEQ ID NO: 1) HLA-A*02 complex and not to apanel of alternative peptide-HLA complexes. This may, for example, bedetermined by the Biacore method of Example 3. Said panel may contain atleast 5, and preferably at least 10, alternative peptide-HLA-A*02complexes. The alternative peptides may share a low level of sequenceidentity with SEQ ID NO: 1 and may be naturally presented. Alternativepeptides are preferably derived from proteins expressed in healthy humantissues. Binding of the TCR to the SLLQHLIGL-HLA-A*02 complex(“SLLQHLIGL” disclosed as SEQ ID NO: 1) may be at least 2 fold greaterthan to other naturally-presented peptide HLA complexes, more preferablyat least 10 fold, or at least 50 fold or at least 100 fold greater, evenmore preferably at least 400 fold greater.

An alternative or additional approach to determine TCR specificity maybe to identify the peptide recognition motif of the TCR using sequentialmutagenesis, e.g. alanine scanning. Residues that form part of thebinding motif are those that are not permissible to substitution.Non-permissible substitutions may be defined as those peptide positionsin which the binding affinity of the TCR is reduced by at least 50%, orpreferably at least 80% relative to the binding affinity for thenon-mutated peptide. Such an approach is further described in Cameron etal., (2013), Sci Transl Med. 2013 Aug. 7; 5 (197): 197ra103 andWO2014096803. TCR specificity in this case may be determined byidentifying alternative motif containing peptides, particularlyalternative motif containing peptides in the human proteome, and testingthese peptides for binding to the TCR. Binding of the TCR to one or morealternative peptides may indicate a lack of specificity. In this casefurther testing of TCR specificity via cellular assays may be required.

TCRs of the invention may have an ideal safety profile for use astherapeutic reagents. In this case the TCRs may be in soluble form andmay preferably be fused to an immune effector. Suitable immune effectorsinclude but are not limited to, cytokines, such as IL-2 and IFN-γ;superantigens and mutants thereof; chemokines such as IL-8, plateletfactor 4, melanoma growth stimulatory protein; antibodies, includingfragments, derivatives and variants thereof, that bind to antigens onimmune cells such as T cells or NK cell (e.g. anti-CD3, anti-CD28 oranti-CD16); and complement activators. An ideal safety profile meansthat in addition to demonstrating good specificity, the TCRs of theinvention may have passed further preclinical safety tests. Examples ofsuch tests include whole blood assays to confirm minimal cytokinerelease in the presence of whole blood and thus low risk of causing apotential cytokine release syndrome in vivo, and alloreactivity tests toconfirm low potential for recognition of alternative HLA types.

TCRs of the invention may be amenable to high yield purification,particularly TCRs in soluble format. Yield may be determined based onthe amount of material retained during the purification process (i.e.the amount of correctly folded material obtained at the end of thepurification process relative to the amount of solubilised materialobtained prior to refolding), and or yield may be based on the amount ofcorrectly folded material obtained at the end of the purificationprocess, relative to the original culture volume. High yield meansgreater than 1%, or more preferably greater than 5%, or higher yield.High yield means greater than 1 mg/ml, or more preferably greater than 3mg/ml, or greater than 5 mg/ml, or higher yield.

TCRs of the invention preferably have a K_(D) for the SLLQHLIGL-HLA-A*02complex (“SLLQHLIGL” disclosed as SEQ ID NO: 1) of greater than (i.e.stronger than) the non-mutated, or scaffold TCR, for example in therange of 1 μM to 100 μM. In one aspect, TCRs of the invention have aK_(D) for the complex of from about (i.e. +/−10%) 1 μM to about 400 nM,from about 1 μM to about 1000 μM, from about 1 μM to about 500 μM. SaidTCRs may additionally, or alternatively, have a binding half-life (T½)for the complex in the range of from about 1 min to about 60 h, fromabout 20 min to about 50 h, or from about 2 h to about 35 h. In aparticularly preferred embodiment, TCRs of the invention have a K_(D)for the SLLQHLIGL-HLA-A*02 complex (“SLLQHLIGL” disclosed as SEQ IDNO: 1) of from about 1 μM to about 500 μM and/or a binding half-lifefrom about 2 h to about 35 h. Such high-affinity is preferable for TCRsin soluble format when associated with therapeutic agents and/ordetectable labels.

In another aspect, TCRs of the invention may have a K_(D) for thecomplex of from about 50 nM to about 200 μM, or from about 100 nM toabout 1 μM and/or a binding half-life for the complex of from about 3sec to about 12 min. Such TCRs may be preferable for adoptive therapyapplications.

Methods to determine binding affinity (inversely proportional to theequilibrium constant K_(D)) and binding half life (expressed as T½) areknown to those skilled in the art. In a preferred embodiment, bindingaffinity and binding half-life are determined using Surface PlasmonResonance (SPR) or Bio-Layer Interferometry (BLI), for example using aBIAcore instrument or Octet instrument, respectively. A preferred methodis provided in Example 3. It will be appreciated that doubling theaffinity of a TCR results in halving the K_(D). T½ is calculated as In2divided by the off-rate (k_(off)). Therefore, doubling of T½ results ina halving in k_(off). K_(D) and k_(off) values for TCRs are usuallymeasured for soluble forms of the TCR, i.e. those forms which aretruncated to remove cytoplasmic and transmembrane domain residues. Toaccount for variation between independent measurements, and particularlyfor interactions with dissociation times in excess of 20 hours, thebinding affinity and or binding half-life of a given TCR may be measuredseveral times, for example 3 or more times, using the same assayprotocol, and an average of the results taken. To compare binding databetween two samples (i.e. two different TCRs and or two preparations ofthe same TCR) it is preferable that measurements are made using the sameassay conditions (e.g. temperature), such as those described in Example3.

Certain preferred TCRs of the invention have a binding affinity for,and/or a binding half-life for, the SLLQHLIGL-HLA-A*02 complex(“SLLQHLIGL” disclosed as SEQ ID NO: 1) that is substantially higherthan that of the native TCR. Increasing the binding affinity of a nativeTCR often reduces the specificity of the TCR for its peptide-MHC ligand,and this is demonstrated in Zhao et al., (2007) J. Immunol, 179:9,5845-5854. However, such TCRs of the invention remain specific for theSLLQHLIGL-HLA-A*02 complex (“SLLQHLIGL” disclosed as SEQ ID NO: 1),despite having substantially higher binding affinity than the nativeTCR.

Certain preferred TCRs are able to generate a highly potent T cellresponse in vitro against antigen positive cells, in particular thosecells presenting low levels of antigen typical of cancer cells (i.e. inthe order of 5-100, for example 50, antigens per cell (Bossi et al.,(2013) Oncoimmunol. 1; 2 (11) :e26840; Purbhoo et al., (2006). J Immunol176(12): 7308-7316.)). Such TCRs may be in soluble form and linked to animmune effector such as an anti-CD3 antibody. The T cell response thatis measured may be the release of T cell activation markers such asInterferon γ or Granzyme B, or target cell killing, or other measure ofT cell activation, such as T cell proliferation. Preferably a highlypotent response is one with EC₅₀ value in the pM range, most preferably,100 μM or lower.

TCRs of the invention may be αβ heterodimers. Alpha-beta heterodimericTCRs of the invention usually comprise an alpha chain TRAC constantdomain sequence and/or a beta chain TRBC1 or TRBC2 constant domainsequence. The constant domains may be full-length by which it is meantthat extracellular, transmembrane and cytoplasmic domains are present,or they may be in soluble format (i.e. having no transmembrane orcytoplasmic domains). One or both of the constant domains may containmutations, substitutions or deletions relative to the native TRAC and/orTRBC1/2 sequences. The term TRAC and TRBC1/2 also encompasses naturalpolymorphic variants, for example N to K at position 4 of TRAC (Bragadoet al International immunology. 1994 February; 6(2):223-30).

For soluble TCRs of the invention, the alpha and beta chain constantdomain sequences may be modified by truncation or substitution to deletethe native disulphide bond between Cys4 of exon 2 of TRAC and Cys2 ofexon 2 of TRBC1 or TRBC2. The alpha and/or beta chain constant domainsequence(s) may have an introduced disulphide bond between residues ofthe respective constant domains, as described, for example, in WO03/020763. In a preferred embodiment the alpha and beta constant domainsmay be modified by substitution of cysteine residues at position Thr 48of TRAC and position Ser 57 of TRBC1 or TRBC2, the said cysteinesforming a disulphide bond between the alpha and beta constant domains ofthe TCR. TRBC1 or TRBC2 may additionally include a cysteine to alaninemutation at position 75 of the constant domain and an asparagine toaspartic acid mutation at position 89 of the constant domain. One orboth of the extracellular constant domains present in an αβ heterodimerof the invention may be truncated at the C terminus or C termini, forexample by up to 15, or up to 10, or up to 8 or fewer amino acids. Oneor both of the extracellular constant domains present in an αβheterodimer of the invention may be truncated at the C terminus or Ctermini by, for example, up to 15, or up to 10 or up to 8 amino acids.The C terminus of the alpha chain extracellular constant domain may betruncated by 8 amino acids. Soluble TCRs are preferably associated withtherapeutic agents and/or detectable labels

The constant domains of an αβ heterodimeric TCR may be full length,having both transmembrane and cytoplasmic domains. Such TCRs may containa disulphide bond corresponding to that found in nature between therespective alpha and beta constant domains. Additionally, oralternatively, a non-native disulphide bond may be present between theextracellular constant domains. Said non-native disulphide bonds arefurther described in WO03020763 and WO06000830. The non-nativedisulphide bond may be between position Thr 48 of TRAC and position Ser57 of TRBC1 or TRBC2. One or both of the constant domains may containone or more mutations substitutions or deletions relative to the nativeTRAC and/or TRBC1/2 sequences. TCRs with full-length constant domainsare preferable for use in adoptive therapy.

TCRs of the invention may be in single chain format. Single chainformats include, but are not limited to, αβ TCR polypeptides of theVα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, or Vα-Cα-L-Vβ-Cβ types,wherein Vα and Vβ are TCR α and β variable regions respectively, Cα andCβ are TCR α and β constant regions respectively, and L is a linkersequence (Weidanz et al., (1998) J Immunol Methods. Dec. 1; 221(1-2):59-76; Epel et al., (2002), Cancer Immunol Immunother. November;51(10):565-73; WO 2004/033685; WO9918129). Where present, one or both ofthe constant domains may be full length, or they may be truncated and/orcontain mutations as described above. Preferably single chain TCRs aresoluble. In certain embodiments single chain TCRs of the invention mayhave an introduced disulphide bond between residues of the respectiveconstant domains, as described in WO 2004/033685. Single chain TCRs arefurther described in WO2004/033685; WO98/39482; WO01/62908; Weidanz etal. (1998) J Immunol Methods 221(1-2): 59-76; Hoo et al. (1992) ProcNatl Acad Sci USA 89(10): 4759-4763; Schodin (1996) Mol Immunol 33(9):819-829).

The invention also includes particles displaying TCRs of the inventionand the inclusion of said particles within a library of particles. Suchparticles include but are not limited to phage, yeast cells, ribosomes,or mammalian cells. Method of producing such particles and libraries areknown in the art (for example see WO2004/044004; WO01/48145, Chervin etal. (2008) J. Immuno. Methods 339.2: 175-184).

Soluble TCRs of the invention are useful for delivering detectablelabels or therapeutic agents to antigen presenting cells and tissuescontaining antigen presenting cells. They may therefore be associated(covalently or otherwise) with a detectable label (for diagnosticpurposes wherein the TCR is used to detect the presence of cellspresenting the cognate antigen); and or a therapeutic agent; and or a PKmodifying moiety.

Examples of PK modifying moieties include, but are not limited to, PEG(Dozier et al., (2015) Int J Mol Sci. October 28; 16(10):25831-64 andJevsevar et al., (2010) Biotechnol J. January; 5(1):113-28), PASylation(Schlapschy et al., (2013) Protein Eng Des Sel. August; 26(8):489-501),albumin, and albumin binding domains, (Dennis et al., (2002) J BiolChem. September 20; 277(38):35035-43), and/or unstructured polypeptides(Schellenberger et al., (2009) Nat Biotechnol. December;27(12):1186-90). Further PK modifying moieties include antibody Fcfragments.

Detectable labels for diagnostic purposes include for instance,fluorescent labels, radiolabels, enzymes, nucleic acid probes andcontrast reagents.

For some purposes, the TCRs of the invention may be aggregated into acomplex comprising several TCRs to form a multivalent TCR complex. Thereare a number of human proteins that contain a multimerisation domainthat may be used in the production of multivalent TCR complexes. Forexample the tetramerisation domain of p53 which has been utilised toproduce tetramers of scFv antibody fragments which exhibited increasedserum persistence and significantly reduced off-rate compared to themonomeric scFv fragment (Willuda et al. (2001) J. Biol. Chem. 276 (17)14385-14392). Haemoglobin also has a tetramerisation domain that couldbe used for this kind of application. A multivalent TCR complex of theinvention may have enhanced binding capability for the complex comparedto a non-multimeric wild-type or T cell receptor heterodimer of theinvention.

Thus, multivalent complexes of TCRs of the invention are also includedwithin the invention. Such multivalent TCR complexes according to theinvention are particularly useful for tracking or targeting cellspresenting particular antigens in vitro or in vivo, and are also usefulas intermediates for the production of further multivalent TCR complexeshaving such uses.

Therapeutic agents which may be associated with the TCRs of theinvention include immune-modulators and effectors, radioactivecompounds, enzymes (perforin for example) or chemotherapeutic agents(cis-platin for example). To ensure that toxic effects are exercised inthe desired location the toxin could be inside a liposome linked to TCRso that the compound is released slowly. This will prevent damagingeffects during the transport in the body and ensure that the toxin hasmaximum effect after binding of the TCR to the relevant antigenpresenting cells.

Examples of suitable therapeutic agents include, but are not limited to:

-   -   small molecule cytotoxic agents, i.e. compounds with the ability        to kill mammalian cells having a molecular weight of less than        700 Daltons. Such compounds could also contain toxic metals        capable of having a cytotoxic effect. Furthermore, it is to be        understood that these small molecule cytotoxic agents also        include pro-drugs, i.e. compounds that decay or are converted        under physiological conditions to release cytotoxic agents.        Examples of such agents include cis-platin, maytansine        derivatives, rachelmycin, calicheamicin, docetaxel, etoposide,        gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone,        sorfimer sodiumphotofrin II, temozolomide, topotecan,        trimetreate 21arbour2late, auristatin E vincristine and        doxorubicin;    -   peptide cytotoxins, i.e. proteins or fragments thereof with the        ability to kill mammalian cells. For example, ricin, diphtheria        toxin, pseudomonas bacterial exotoxin A, Dnase and Rnase;    -   radio-nuclides, i.e. unstable isotopes of elements which decay        with the concurrent emission of one or more of α or β particles,        or γ rays. For example, iodine 131, rhenium 186, indium 111,        yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213;        chelating agents may be used to facilitate the association of        these radio-nuclides to the high affinity TCRs, or multimers        thereof;    -   Immuno-stimulants, i.e. immune effector molecules which        stimulate immune response. For example, cytokines such as IL-2        and IFN-γ,    -   Superantigens and mutants thereof;    -   TCR-HLA fusions, e.g. fusion to a peptide-HLA complex, wherein        said peptide is derived from a common human pathogen, such as        Epstein Barr Virus (EBV);    -   chemokines such as IL-8, platelet factor 4, melanoma growth        stimulatory protein, etc;    -   antibodies or fragments thereof, including anti-T cell or NK        cell determinant antibodies (e.g. anti-CD3, anti-CD28 or        anti-CD16);    -   alternative protein scaffolds with antibody like binding        characteristics    -   complement activators;    -   xenogeneic protein domains, allogeneic protein domains,        viral/bacterial protein domains, viral/bacterial peptides.

One preferred embodiment is provided by a soluble TCR of the inventionassociated (usually by fusion to the N- or C-terminus of the alpha orbeta chain) with an immune effector. A particularly preferred immuneeffector is an anti-CD3 antibody, or a functional fragment or variant ofsaid anti-CD3 antibody (such TCR-anti-CD3 fusions may be termed ImmTAC™molecules). As used herein, the term “antibody” encompasses suchfragments and variants. Examples of anti-CD3 antibodies include but arenot limited to OKT3, UCHT-1, BMA-031 and 12F6. Antibody fragments andvariants/analogues which are suitable for use in the compositions andmethods described herein include minibodies, Fab fragments, F(ab′)₂fragments, dsFv and scFv fragments, Nanobodies™ (these constructs,marketed by Ablynx (Belgium), comprise synthetic single immunoglobulinvariable heavy domain derived from a camelid (e.g. camel or llama)antibody) and Domain Antibodies (Domantis (Belgium), comprising anaffinity matured single immunoglobulin variable heavy domain orimmunoglobulin variable light domain) or alternative protein scaffoldsthat exhibit antibody like binding characteristics such as Affibodies(Affibody (Sweden), comprising engineered protein A scaffold) orAnticalins (Pieris (Germany)), comprising engineered anticalins) to namebut a few.

Linkage of the TCR and the anti-CD3 antibody may be via covalent ornon-covalent attachment. Covalent attachment may be direct, or indirectvia a linker sequence. Linker sequences are usually flexible, in thatthey are made up primarily of amino acids such as glycine, alanine andserine, which do not have bulky side chains likely to restrictflexibility. Alternatively, linkers with greater rigidity may bedesirable. Usable or optimum lengths of linker sequences may be easilydetermined. Often the linker sequence will be less than about 12, suchas less than 10, or from 2-10 amino acids in length. Examples ofsuitable linkers that may be used in TCRs of the invention include, butare not limited to: GGGGS (SEQ ID NO: 31), GGGSG (SEQ ID NO: 32), GGSGG(SEQ ID NO: 33), GSGGG (SEQ ID NO: 34), GSGGGP (SEQ ID NO: 35), GGEPS(SEQ ID NO: 36), GGEGGGP (SEQ ID NO: 37), and GGEGGGSEGGGS (SEQ ID NO:38) (as described in WO2010/133828).

Specific embodiments of anti-CD3-TCR fusion constructs of the inventioninclude those alpha and beta chain pairings in which the alpha chain iscomposed of a TCR variable domain comprising the amino acid sequence ofSEQ ID NOs: 6-8 and/or the beta chain is composed of a TCR variabledomain comprising the amino acid sequence of SEQ ID NOs: 9-24. Saidalpha and beta chains may further comprise a constant region comprisinga non-native disulphide bond. The constant domain of the alpha chain maybe truncated by eight amino acids. The N or C terminus of the alpha andor beta chain may be fused to an anti-CD3 scFv antibody fragment via alinker selected from SEQ ID NOs: 31-38. Certain preferred embodiments ofsuch anti-CD3-TCR fusion constructs are provided below:

Alpha chain Beta Chain SEQ ID NO SEQ ID NO SEQ ID NO 25 SEQ ID NO 26 SEQID NO 27 SEQ ID NO 28 SEQ ID NO 29 SEQ ID NO 30

Also included within the scope of the invention are functional variantsof said anti-CD3-TCR fusion constructs. Said functional variantspreferably have at least 90% identity, such as 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identity to the reference sequence, butare nonetheless functionally equivalent.

In a further aspect, the present invention provides nucleic acidencoding a TCR, or TCR anti-CD3 fusion of the invention. In someembodiments, the nucleic acid is cDNA. In some embodiments the nucleicacid may be mRNA. In some embodiments, the invention provides nucleicacid comprising a sequence encoding an α chain variable domain of a TCRof the invention. In some embodiments, the invention provides nucleicacid comprising a sequence encoding a β chain variable domain of a TCRof the invention. The nucleic acid may be non-naturally occurring and/orpurified and/or engineered. The nucleic acid sequence may be codonoptimised, in accordance with expression system utilised. As is known tothose skilled in the art, expression systems may include bacterial cellssuch as E. coli, or yeast cells, or mammalian cells, or insect cells, orthey may be cell free expression systems.

In another aspect, the invention provides a vector which comprisesnucleic acid of the invention. Preferably the vector is a TCR expressionvector. Suitable TCR expression vectors include, for example,gamma-retroviral vectors or, more preferably, lentiviral vectors.Further details can be found in Zhang 2012 and references therein (Zhanget al., Adv Drug Deliv Rev. 2012 Jun. 1; 64(8): 756-762).

The invention also provides a cell harbouring a vector of the invention,preferably a TCR expression vector. Suitable cells include, mammaliancells, preferably immune cells, even more preferably T cells. The vectormay comprise nucleic acid of the invention encoding in a single openreading frame, or two distinct open reading frames, encoding the alphachain and the beta chain respectively. Another aspect provides a cellharbouring a first expression vector which comprises nucleic acidencoding the alpha chain of a TCR of the invention, and a secondexpression vector which comprises nucleic acid encoding the beta chainof a TCR of the invention. Such cells are particularly useful inadoptive therapy. The cells of the invention may be isolated and/orrecombinant and/or non-naturally occurring and/or engineered.

Since the TCRs of the invention have utility in adoptive therapy, theinvention includes a non-naturally occurring and/or purified and/or orengineered cell, especially a T-cell, presenting a TCR of the invention.The invention also provides an expanded population of T cells presentinga TCR of the invention. There are a number of methods suitable for thetransfection of T cells with nucleic acid (such as DNA, cDNA or RNA)encoding the TCRs of the invention (see for example Robbins et al.,(2008) J Immunol. 180: 6116-6131). T cells expressing the TCRs of theinvention will be suitable for use in adoptive therapy-based treatmentof cancer. As will be known to those skilled in the art, there are anumber of suitable methods by which adoptive therapy can be carried out(see for example Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308).

As is well-known in the art, TCRs may be subject to post translationalmodifications. Glycosylation is one such modification, which comprisesthe covalent attachment of oligosaccharide moieties to defined aminoacids in the TCR chain. For example, asparagine residues, orserine/threonine residues are well-known locations for oligosaccharideattachment. The glycosylation status of a particular protein depends ona number of factors, including protein sequence, protein conformationand the availability of certain enzymes. Furthermore, glycosylationstatus (i.e. oligosaccharide type, covalent linkage and total number ofattachments) can influence protein function. Therefore, when producingrecombinant proteins, controlling glycosylation is often desirable.Controlled glycosylation has been used to improve antibody basedtherapeutics. (Jefferis et al., (2009) Nat Rev Drug Discov March;8(3):226-34.). For soluble TCRs of the invention glycosylation may becontrolled, by using particular cell lines for example (including butnot limited to mammalian cell lines such as Chinese hamster ovary (CHO)cells or human embryonic kidney (HEK) cells), or by chemicalmodification. Such modifications may be desirable, since glycosylationcan improve pharmacokinetics, reduce immunogenicity and more closelymimic a native human protein (Sinclair and Elliott, (2005) Pharm Sci.August; 94(8):1626-35).

For administration to patients, the TCRs of the invention (preferablyassociated with a detectable label or therapeutic agent or expressed ona transfected T cell), TCR-anti CD3 fusion molecules, nucleic acids,expression vectors or cells of the invention may be provided as part ofa sterile pharmaceutical composition together with one or morepharmaceutically acceptable carriers or excipients. This pharmaceuticalcomposition may be in any suitable form, (depending upon the desiredmethod of administering it to a patient). It may be provided in unitdosage form, will generally be provided in a sealed container and may beprovided as part of a kit. Such a kit would normally (although notnecessarily) include instructions for use. It may include a plurality ofsaid unit dosage forms.

The pharmaceutical composition may be adapted for administration by anyappropriate route, such as parenteral (including subcutaneous,intramuscular, intrathecal or intravenous), enteral (including oral orrectal), inhalation or intranasal routes. Such compositions may beprepared by any method known in the art of pharmacy, for example bymixing the active ingredient with the carrier(s) or excipient(s) understerile conditions.

Dosages of the substances of the present invention can vary between widelimits, depending upon the disease or disorder to be treated, the ageand condition of the individual to be treated, etc. a suitable doserange for a TCR-anti-CD3 fusion molecules may be in the range of 25ng/kg to 50 pg/kg or 1 pg to 1 g. A physician will ultimately determineappropriate dosages to be used.

TCRs, TCR-anti-CD3 fusion molecules, pharmaceutical compositions,vectors, nucleic acids and cells of the invention may be provided insubstantially pure form, for example, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% pure.

Also provided by the invention are:

-   -   A TCR, TCR-anti-CD3 fusion molecule, nucleic acid,        pharmaceutical composition or cell of the invention for use in        medicine, preferably for use in a method of treating cancer or a        tumour;    -   the use of a TCR, TCR-anti-CD3 fusion molecule, nucleic acid,        pharmaceutical composition or cell of the invention in the        manufacture of a medicament for treating cancer or a tumour;    -   a method of treating cancer or a tumour in a patient, comprising        administering to the patient a TCR, TCR-anti-CD3 fusion        molecule, nucleic acid, pharmaceutical composition or cell of        the invention;    -   an injectable formulation for administering to a human subject        comprising a TCR, TCR-anti-CD3 fusion molecule, nucleic acid,        pharmaceutical composition or cell of the invention.

The cancer may be a solid or liquid tumour. Preferable the tumourexpresses PRAME. The cancer may be of the breast (including triplenegative), ovary, endometrium, oesophagus, lung (NSCLC and SCLC),bladder or the head and neck. Alternatively or additionally the cancermay be a leukemia or lymphoma. Of these cancers, breast (includingtriple negative), ovary and endometrium are preferred. The TCR,TCR-anti-CD3 fusion molecule, nucleic acid, pharmaceutical compositionor cell of the invention may be administered by injection, such asintravenous or direct intratumoral injection. The human subject may beof the HLA-A*02 subtype.

The method of treatment may further include administering separately, incombination, or sequentially, an additional anti-neoplastic agent.Example of such agents are known in the art and may include immuneactivating agents and/or T cell modulating agents.

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis. The prior art documents mentionedherein are incorporated by reference to the fullest extent permitted bylaw.

DESCRIPTION OF THE DRAWINGS

FIG. 1—provides the amino acid sequence of the extracellular regions ofthe scaffold PRAME TCR alpha and beta chain.

FIG. 2—provides the amino acid sequence of the extracellular regions ofa soluble version of the scaffold PRAME TCR alpha and beta chain.

FIG. 3—provides example amino acid sequences of mutated PRAME TCR alphachain variable regions.

FIG. 4—provides example amino acid sequences of mutated PRAME TCR betachain variable regions.

FIG. 5—provides amino acid sequences of ImmTAC molecules (TCR-anti-CD3fusions) comprising certain mutated PRAME TCR variable domains as setout in FIGS. 3 and 4.

FIG. 6—provides cellular data demonstrating potency and specificity ofImmTAC molecules of FIG. 5 comprising the mutated PRAME TCR variabledomains as set out in FIGS. 3 and 4.

FIG. 7 (panels a and b)—provide cellular data demonstrating specificityof ImmTAC molecules of FIG. 5, comprising the mutated PRAME TCR variabledomains as set out in FIGS. 3 and 4.

FIG. 8—provides cellular data demonstrating killing of PRAME positivemelanoma cancer cells by ImmTAC molecules of FIG. 5, comprising themutated PRAME TCR variable domains as set out in FIGS. 3 and 4.

FIG. 9—provides cellular data demonstrating killing of PRAME positivelung cancer cells by ImmTAC molecules of FIG. 5, comprising the mutatedPRAME TCR variable domains as set out in FIGS. 3 and 4.

The invention is further described in the following non-limitingexamples.

EXAMPLES Example 1—Expression, Refolding and Purification of SolubleTCRs Method

DNA sequences encoding the alpha and beta extracellular regions ofsoluble TCRs of the invention were cloned separately into pGMT7-basedexpression plasmids using standard methods (as described in Sambrook, etal. Molecular cloning. Vol. 2. (1989) New York: Cold spring harbourlaboratory press). The expression plasmids were transformed separatelyinto E. coli strain Rosetta (BL21 pLysS), or T7 Express, and singleampicillin-resistant colonies were grown at 37° C. in TYP (+ampicillin100 μg/ml) medium to an OD₆₀₀ of ˜0.6-0.8 before inducing proteinexpression with 0.5 mM IPTG. Cells were harvested three hourspost-induction by centrifugation. Cell pellets were lysed with BugBusterprotein extraction reagent (Merck Millipore) according to themanufacturer's instructions. Inclusion body pellets were recovered bycentrifugation. Pellets were washed twice in Triton buffer (50 mMTris-HCl pH 8.1, 0.5% Triton-X100, 100 mM NaCl, 10 mM NaEDTA) andfinally resuspended in detergent free buffer (50 mM Tris-HCl pH 8.1, 100mM NaCl, 10 mM NaEDTA). Inclusion body protein yield was quantified bysolubilising with 6 M guanidine-HCl and measuring 00280. Proteinconcentration was then calculated using the extinction coefficient.Inclusion body purity was measured by solubilising with 8M Urea andloading ˜2 μg onto 4-20% SDS-PAGE under reducing conditions. Purity wasthen estimated or calculated using densitometry software (Chemidoc,Biorad). Inclusion bodies were stored at +4° C. for short term storageand at −20° C. or −70° C. for longer term storage.

For soluble TCR refolding, α and β chain-containing inclusion bodieswere first mixed and diluted into 10 ml solubilisation/denaturationbuffer (6 M Guanidine-hydrochloride, 50 mM Tris HCl pH 8.1, 100 mM NaCl,10 mM EDTA, 20 mM DTT) followed by incubation for 30 min at 37° C.Refolding was then initiated by further dilution into 1 L of refoldbuffer (100 mM Tris pH 8.1, 800 or 1000 mM L-Arginine HCL, 2 mM EDTA, 4M Urea, 10 mM cysteamine hydrochloride and 2.5 mM cystaminedihydrochloride) and the solution mixed well. The refolded mixture wasdialysed against 10 L H₂O for 18-20 hours at 5° C.±3° C. After thistime, the dialysis buffer was twice replaced with 10 mM Tris pH 8.1 (10L) and dialysis continued for another 15 hours. The refold mixture wasthen filtered through 0.45 μm cellulose filters.

Purification of soluble TCRs was initiated by applying the dialysedrefold onto a POROS® 50HQ anion exchange column and eluting boundprotein with a gradient of 0-500 mM NaCl in 20 mM Tris pH 8.1 over 6column volumes using an Akta® Pure (GE Healthcare). Peak TCR fractionswere identified by SDS PAGE before being pooled and concentrated. Theconcentrated sample was then applied to a Superdex® 200 Increase 10/300GL gel filtration column (GE Healthcare) pre-equilibrated in Dulbecco'sPBS buffer. The peak TCR fractions were pooled and concentrated and thefinal yield of purified material calculated.

Example 2—Expression, Refolding and Purification of ImmTAC Molecules(Soluble TCR-Anti CD3 Fusion Molecules) Method

ImmTAC preparation was carried out as described in Example 1, exceptthat the TCR beta chain was fused via a linker to an anti-CD3 singlechain antibody. In addition a cation exchange step was performed duringpurification following the anion exchange. In this case the peakfractions from anion exchange were diluted 20-fold in 20 mM MES (pH6.5),and applied to a POROS® 50HS cation exchange column. Bound protein waseluted with a gradient of 0-500 mM NaCl in 20 mM MES. Peak ImmTACfractions were pooled and adjusted to 50 mM Tris pH 8.1, before beingconcentrated and applied directly to the gel filtration matrix asdescribed in Example 1.

Example 3—Binding Characterisation

Binding analysis of purified soluble TCRs and ImmTAC molecules to therelevant peptide-HLA complex was carried out by surface plasmonresonance, using a BIAcore 3000 or BIAcore T200 instrument, or bybiolayer interferometry, using a ForteBio Octet instrument).Biotinylated class I HLA-A*02 molecules were refolded with the peptideof interest and purified using methods known to those in the art(O'Callaghan et al. (1999). Anal Biochem 266(1): 9-15; Garboczi, et al.(1992). Proc Natl Acad Sci USA 89(8): 3429-3433). All measurements wereperformed at 25° C. in Dulbecco's PBS buffer, supplemented with 0.005%P20.

BIAcore Method

Biotinylated peptide-HLA monomers were immobilized on tostreptavidin-coupled CM-5 sensor chips. Equilibrium binding constantswere determined using serial dilutions of soluble TCR/ImmTAC injected ata constant flow rate of 30 μl min⁻¹ over a flow cell coated with ˜200response units (RU) of peptide-HLA-A*02 complex. Equilibrium responseswere normalised for each TCR concentration by subtracting the bulkbuffer response on a control flow cell containing an irrelevantpeptide-HLA. The K_(D) value was obtained by non-linear curve fittingusing Prism software and the Langmuir binding isotherm,bound=C*Max/(C+K_(D)), where “bound” is the equilibrium binding in RU atinjected TCR concentration C and Max is the maximum binding.

For high affinity interactions, binding parameters were determined bysingle cycle kinetics analysis. Five different concentrations of solubleTCR/ImmTAC were injected over a flow cell coated with ˜100-200 RU ofpeptide-HLA complex using a flow rate of 50-60 μl min⁻¹. Typically,60-120 μl of soluble TCR/ImmTAC was injected at a top concentration ofbetween 50-100 nM, with successive 2 fold dilutions used for the otherfour injections. The lowest concentration was injected first. To measurethe dissociation phase buffer was then injected until 10% dissociationoccurred, typically after 1-3 hours. Kinetic parameters were calculatedusing BIAevaluation® software. The dissociation phase was fitted to asingle exponential decay equation enabling calculation of half-life. Theequilibrium constant K_(D) was calculated from k_(off)/k_(on).

Octet Method

Biotinylated peptide-HLA monomers were captured to 1 nm on to (SA)streptavidin biosensors (Pall ForteBio) pre-immobilised withstreptavidin. The sensors were blocked with free biotin (2 μM) for 2minutes. Equilibrium binding constants were determined by immersing theloaded biosensors into soluble TCR/ImmTAC serially diluted in a 96-wellor 384-well sample plate. Plate shaking was set to 1000 rpm. For lowaffinity interactions (μM range) a short association (˜2 minutes) and ashort dissociation time (˜2 minutes) was used. Binding curves wereprocessed by double reference subtraction of reference biosensors loadedwith irrelevant pHLA using Octet Data Analysis Software (Pall ForteBio).Responses (nm) at equilibrium were used to estimate the K_(D) value fromsteady state plots fitted to the equation Response=Rmax*conc/(KD+conc),where “response” is the equilibrium binding in nm at each TCRconcentration (cone) and Rmax is the maximum binding response at pHLAsaturation.

For high affinity interactions (nM-pM range), kinetic parameters weredetermined from binding curves at 3 TCR/ImmTAC concentrations typically10 nM, 5 nM and 2.5 nM. The association time was 30 minutes and thedissociation time 1-2 hours. Binding curves were processed by doublereference subtraction of reference biosensors loaded with irrelevantpHLA and blocked with biotin. Kinetic parameters k_(on) and k_(off) werecalculated by global fitting directly to the binding curves using OctetData Analysis Software (Pall ForteBio). K_(D) was calculated fromk_(off)/k_(on) and the dissociation half-life was calculated fromt_(1/2)=0.693/k_(off).

Example 4—Binding Characterisation of the Native TCR

A soluble native TCR was prepared according to the methods described inExample 1 and binding to pHLA analysed according to Example 3. The aminoacid sequences of the alpha and beta chains corresponded to those shownin FIG. 2. Soluble biotinylated HLA-A*02 was prepared with the PRAMEpeptide SLLQHLIGL (SEQ ID NO: 1) and immobilised onto a BIAcore sensorchip.

Results

Binding was determined at various concentrations and the K_(D) value forthe interaction was determined to be 141 μM. Cross reactivity(specificity) was assessed against a panel of 14 irrelevant peptideHLA-A*02 complexes using the equilibrium BIAcore method of Example 3.The 14 irrelevant pHLAs were divided into three groups and loaded ontoone of three flow cells, to give approximately 1000 RU of each pHLA perflow cell. 30 μL of soluble wild type TCR was injected at concentrationsof 130 and 488 μM over all flow cells at a rate of 20 μL/min. Nosignificant binding was detected at either concentration indicting thatthe native TCR is specific for the SLLQHLIGL-HLA-A*02 complex(“SLLQHLIGL” disclosed as SEQ ID NO: 1).

These data indicate that this native TCR has characteristics that aresuitable for use as a starting sequence for engineering high affinitytherapeutic TCRs.

Example 5—Binding Characterisation of Certain Mutated TCRs of theInvention

The mutated TCR alpha and beta variable domain amino acid sequences,provided in FIGS. 3 and 4 respectively (SEQ ID NOs: 6-24), were used toprepare ImmTAC molecules. Note that inclusion of a glycine residue atthe start of the alpha chain (˜1 position relative to the numbering ofSEQ ID NO: 2) was found to improve cleavage efficiency of the N terminalmethionine during production in E. coli. Inefficient cleavage may bedetrimental for a therapeutic since it may result in a heterogeneousprotein product and or the presence of the initiation methionine may beimmunogenic in humans. Full amino acid sequences of ImmTAC moleculescomprising the following alpha and beta chains are provided in FIG. 5

-   -   a28b50—ImmTAC1    -   a79674—ImmTAC2    -   a79b46—ImmTAC3

The molecules were prepared as described in Example 2 and binding toSLLQHLIGL-HLA-A*02 complex (“SLLQHLIGL” disclosed as SEQ ID NO: 1) wasdetermined according to Example 3.

Results

The data presented in the table below show that ImmTAC moleculescomprising the indicated TCR variable domain sequences recognisedSLLQHLIGL-HLA-A*02 complex (“SLLQHLIGL” disclosed as SEQ ID NO: 1) witha particularly suitable affinity and/or half-life.

α chain β chain k_(D) t_(1/2) a28 (SEQ ID NO: 6) b50 (SEQ ID NO: 9) 391pM 1.8 h a28 (SEQ ID NO: 6) b60 (SEQ ID NO: 19) 261 pM 2.8 h a28 (SEQ IDNO: 6) b74 (SEQ ID NO: 17) 182 pM 3.7 h a28 (SEQ ID NO: 6) b75 (SEQ IDNO: 20) 214 pM 5.1 h a28 (SEQ ID NO: 6) b57 (SEQ ID NO: 10) 83 pM 8.3 ha28 (SEQ ID NO: 6) b58 (SEQ ID NO: 21) 79 pM 8.9 h a79 (SEQ ID NO: 7)b46 (SEQ ID NO: 11) 31.8 pM 29.2 h a109 (SEQ ID NO: 8) b46 (SEQ ID NO:11) 170 pM 7.31 h a79 (SEQ ID NO: 7) b63 (SEQ ID NO: 22) 79 pM 10.8 ha79 (SEQ ID NO: 7) b64 (SEQ ID NO: 12) 138 pM 6.38 h a79 (SEQ ID NO: 7)b66 (SEQ ID NO: 23) 89 pM 9.16 h a79 (SEQ ID NO: 7) b67 (SEQ ID NO: 13)47 pM 12.69 h a79 (SEQ ID NO: 7) b69 (SEQ ID NO: 14) 52 pM 20.41 h a79(SEQ ID NO: 7) b71 (SEQ ID NO: 15) 87 pM 14.89 h a79 (SEQ ID NO: 7) b58(SEQ ID NO: 21) 23.1 pM 28.7 h a79 (SEQ ID NO: 7) b73 (SEQ ID NO: 16)132 pM 4.6 h a79 (SEQ ID NO: 7) b74 (SEQ ID NO: 17) 53.3 pM 12.5 h a79(SEQ ID NO: 7) b75 (SEQ ID NO: 20) 57.7 pM 16.9 h a79 (SEQ ID NO: 7) b76(SEQ ID NO: 24) 11.8 pM 58.3 h a79 (SEQ ID NO: 7) b77 (SEQ ID NO: 18)77.9 pM 8.6 h

Example 6—Potency and Specificity Characterisation of Certain MutatedTCRs of the Invention

ImmTAC molecules comprising the same TCR variable domain sequences asset out in Example 5 were assessed for their ability to mediate potentand specific redirection of CD3+ T cells against PRAME positive cancercells. Interferon-γ (IFN-γ) release was used as a read out for T cellactivation. Full amino acid sequences of ImmTAC molecules comprising thefollowing alpha and beta chains are provided in FIG. 5

-   -   a28b50—ImmTAC1    -   a79674—ImmTAC2    -   a79b46—ImmTAC3

Assays were performed using a human IFN-γ ELISPOT kit (BD Biosciences)according to the manufacturers instructions. Briefly, target cells wereprepared at a density of 1×10⁶/ml in assay medium (RPMI 1640 containing10% heat inactivated FBS and 1% penicillin-streptomycin-L-glutamine) andplated at 50,000 cells per well in a volume of 50 μl. Peripheral bloodmononuclear cells (PBMC), isolated from fresh donor blood, were used aseffector cells and plated at 50,000 cells per well in a volume of 50 μl(the exact number of cells used for each experiment is donor dependentand may be adjusted to produce a response within a suitable range forthe assay). ImmTAC molecules were titrated to give final concentrationsof 10 nM, 1 nM, 0.1 nM, 0.01 nM and 0.001 nM, spanning the anticipatedclinically relevant range, and added to the well in a volume of 50 μl.

Plates were prepared according to the manufacturer's instructions.Target cells, effector cells and ImmTAC molecules were added to therelevant wells and made up to a final volume of 200 μl with assaymedium. All reactions were performed in triplicate. Control wells werealso prepared with the omission of, ImmTAC, effector cells, or targetcells. The plates were then incubated overnight (37° C./5% CO₂). Thenext day the plates were washed three times with wash buffer (1×PBSsachet, containing 0.05% Tween-20, made up in deionised water). Primarydetection antibody was then added to each well in a volume of 50 μl.Plates were incubated at room temperature for 2 hours prior to beingwashed again three times. Secondary detection was performed by adding 50μl of diluted streptavidin-HRP to each well and incubating at roomtemperature for 1 hour and the washing step repeated. No more than 15mins prior to use, one drop (20 μl) of AEC chromogen was added to each 1ml of AEC substrate and mixed and 50 μl added to each well. Spotdevelopment was monitored regularly and plates were washed in tap waterto terminate the development reaction. The plates were then allowed todry at room temperature for at least 2 hours prior to counting the spotsusing a CTL analyser with Immunospot software (Cellular TechnologyLimited).

In this example, the following cancer cells lines were used as targetcells:

Mel624 (melanoma) PRAME + ve HLA-A*02 + ve Granta519 (hemo-lymphocytic)PRAME − ve HLA-A*02 + ve SW620 (colon carcinoma) PRAME − ve HLA-A*02 +ve HT144 (melanoma) PRAME + ve HLA-A*02 − ve

Results

Each of the ImmTAC molecules, comprising the alpha and beta variabledomains indicated in the table below, demonstrated potent activation ofredirected T cells in the presence of antigen positive Mel624 cells. Ineach case, EC50 values were calculated from the data and are shown inthe table below. In addition, each ImmTAC molecule demonstrated minimalor no recognition of two antigen negative, HLA-A*02 positive cells, at aconcentration of up to 1 nM. The ImmTAC molecules also demonstrated norecognition of PRAME positive cells that are HLA-A*02 negative (data notshown). FIG. 6 shows representative data from four of the ImmTACmolecules listed in the table below.

α chain β chain EC₅₀ (SEQ ID NO) (SEQ ID NO) (mel624) a28 (SEQ ID NO: 6)b50 (SEQ ID NO: 9) 34.8 pM a28 (SEQ ID NO: 6) b60 (SEQ ID NO: 19) 31.7pM a28 (SEQ ID NO: 6) b74 (SEQ ID NO: 17) 24.3 pM a28 (SEQ ID NO: 6) b75(SEQ ID NO: 20) 13.9 pM a28 (SEQ ID NO: 6) b57 (SEQ ID NO: 10) 13.4 pMa28 (SEQ ID NO: 6) b58 (SEQ ID NO: 21) 12 pM a79 (SEQ ID NO: 7) b46 (SEQID NO: 11) 18.6 pM a109 (SEQ ID NO: 8) b46 (SEQ ID NO: 11) 60.1 pM a79(SEQ ID NO: 7) b63 (SEQ ID NO: 22) 22.9 pM a79 (SEQ ID NO: 7) b64 (SEQID NO: 12) 27.5 pM a79 (SEQ ID NO: 7) b66 (SEQ ID NO: 23) 16.7 pM a79(SEQ ID NO: 7) b67 (SEQ ID NO: 13) 26.3 pM a79 (SEQ ID NO: 7) b69 (SEQID NO: 14) 39.8 pM a79 (SEQ ID NO: 7) b71 (SEQ ID NO: 15) 31.8 pM a79(SEQ ID NO: 7) b58 (SEQ ID NO: 21) 10.6 pM a79 (SEQ ID NO: 7) b73 (SEQID NO: 16) 23.1 pM a79 (SEQ ID NO: 7) b74 (SEQ ID NO: 17) 9.55 pM a79(SEQ ID NO: 7) b75 (SEQ ID NO: 20) 23.6 pM a79 (SEQ ID NO: 7) b76 (SEQID NO: 24) 17.2 pM a79 (SEQ ID NO: 7) b77 (SEQ ID NO: 18) 13.8 pM

These data demonstrate that ImmTAC molecules comprising mutated TCRvariable domain sequences of the invention can mediate potent andspecific T cell redirection against PRAME positive, HLA-A*02 positive,cancer cells, in a concentration range suitable for therapeutic use.

Example 7—Further Specificity Characterisation of Certain Mutated TCRsof the Invention

To further demonstrate the specificity of ImmTAC molecules comprisingthe mutated TCR sequences, further testing was carried out using thesame ELISPOT methodology as described in Example 6, with a panel ofnormal cells derived from healthy human tissues as target cells.

-   -   Normal tissues included cardiovascular, renal, skeletal muscle,        pulmonary, vasculature, hepatic and brain. In each case antigen        positive Mel624 cancer cells were used as a positive control.

The data presented in this example includes ImmTAC molecules comprisingthe following TCR alpha and beta chains

-   -   a28b50    -   a79674    -   a79b46    -   a79677

The full amino acid sequences of ImmTAC molecules comprising a28b50,a79674 and a79b46 are provided in FIG. 5 (ImmTAC 1-3 respectively)

Results

The data presented in FIG. 7 (panel a) demonstrate that ImmTAC moleculescomprising mutated alpha and beta chain a28b50 and a79b46 show minimalreactivity against a panel of 8 normal cells relative to antigenpositive cancer cells at a concentration up to 1 nM. Likewise, the datain FIG. 7 (panel b) demonstrate that ImmTAC molecules comprising a28b57and a79b46 show minimal reactivity against a panel of 4 normal cellsrelative to antigen positive cancer cells at a concentration up to 1 nM.

Example 8—Cancer Cell Killing Mediated by Certain Mutated TCRs of theInvention

The ability of ImmTAC molecules comprising the mutated TCR sequences tomediate potent redirected T cell killing of antigen positive tumourcells was investigated using the IncuCyte platform (Essen BioScience).This assay allows real time detection by microscopy of the release ofCaspase-3/7, a marker for apoptosis.

Method

Assays were performed using the CellPlayer 96-well Caspase-3/7 apoptosisassay kit (Essen BioScience, Cat. No. 4440) and carried out accordingthe manufacturers protocol. Briefly, target cells (Mel624 (PRAME+veHLA-A*02+ve) or NCI-H1755) were plated at 10,000 cells per well andincubated overnight to allow them to adhere. ImmTAC molecules wereprepared at various concentrations and 25 μl of each was added to therelevant well such that final concentrations were between 1 μM and 100μM. Effector cells were used at an effector target cell ratio of 10:1(100,000 cells per well). A control sample without ImmTAC was alsoprepared along with samples containing either effector cells alone, ortarget cells alone. NucView assay reagent was made up at 30 μM and 25 μladded to every well and the final volume brought to 150 μl (giving 5 μMfinal cone). The plate was placed in the IncuCyte instrument and imagestaken every 2 hours (1 image per well) over 3 days. The number ofapoptotic cells in each image was determined and recorded as apoptoticcells per mm². Assays were performed in triplicate.

The data presented in this example includes ImmTAC molecules comprisingthe following TCR alpha and beta chains

-   -   a28b50    -   a79674    -   a79b46

The full amino acid sequences of ImmTAC molecules comprising a28b50,a79674 and a79b46 are provided in FIG. 5 (ImmTAC 1, 2 and 3respectively).

Results

The data presented in FIGS. 8 and 9 shows real-time killing of antigenpositive cancer cells (Melanoma cell lines Mel624 in FIG. 8 and Lungcancer cell line NCI-H1755 in FIG. 9) in the presence of ImmTACmolecules comprising the mutated TCR sequences, at a concentration of100 pM or lower. No killing was observed in the absence of ImmTACmolecules.

1. A heterodimeric TCR-anti-CD3 antibody fusion molecule, wherein thealpha chain of the fusion molecule has the amino acid sequence set forthin SEQ ID NO: 25 and the beta chain of the fusion molecule has the aminoacid sequence set forth in SEQ ID NO: 26; the alpha chain of the fusionmolecule has the amino acid sequence set forth in SEQ ID NO: 27 and thebeta chain of the fusion molecule has the amino acid sequence set forthin SEQ ID NO: 28; or the alpha chain of the fusion molecule has theamino acid sequence set forth in SEQ ID NO: 29 and the beta chain of thefusion molecule has the amino acid sequence set forth in SEQ ID NO: 30.2. The heterodimeric TCR-anti-CD3 antibody fusion molecule of claim 1,wherein the alpha chain of the fusion molecule has the amino acidsequence set forth in SEQ ID NO: 25 and the beta chain of the fusionmolecule has the amino acid sequence set forth in SEQ ID NO:
 26. 3. Theheterodimeric TCR-anti-CD3 antibody fusion molecule of claim 1, whereinthe alpha chain of the fusion molecule has the amino acid sequence setforth in SEQ ID NO: 27 and the beta chain of the fusion molecule has theamino acid sequence set forth in SEQ ID NO:
 28. 4. The heterodimericTCR-anti-CD3 antibody fusion molecule of claim 1, wherein the alphachain of the fusion molecule has the amino acid sequence set forth inSEQ ID NO: 29 and the beta chain of the fusion molecule has the aminoacid sequence set forth in SEQ ID NO:
 30. 5. A pharmaceuticalcomposition comprising the heterodimeric TCR-anti-CD3 fusion molecule ofclaim 1, together with one or more pharmaceutically acceptable carriersor excipients.
 6. A method of treating a human subject having cancer,comprising: administering to the subject a therapeutically effectivedose of the pharmaceutical composition of claim
 5. 7. The method ofclaim 6, wherein the cancer expresses the antigen PRAME.
 8. The methodof claim 7, wherein the cancer is selected from melanoma, lung cancer,breast cancer, ovarian cancer, endometrial cancer, esophageal cancer,bladder cancer, and head and neck cancer.
 9. The method of claim 8,wherein the lung cancer is non-small cell lung cancer or small cell lungcancer.
 10. The method of claim 8, wherein the breast cancer is triplenegative breast cancer.
 11. The method of claim 6, wherein thepharmaceutical composition is administered by injection.